Bio 2290G Winter/2013 Zabulionis Unit Rm 330 NCB
Session 1: Welcome: Transformation of E. coli
Welcome to the first lab session of the Zabulionis Unit of Biology 2290F—Scientific Methods in Biology.
Beginnings are very important; during this session we’ll get to know one another, and have a look at what is
in store for the 6 sessions in this unit.
It is much better to be proactive than reactive — read, understand, and be prepared for each session.
In the laboratory sessions following the first two, you will design and carry out experiments to answer a
question of your choice regarding the biology of bacteria and plasmids. Some of the biological context for
your experiments will be presented today. This laboratory session also introduces some conceptual
background and technical skills through exercises involving transformation of E. coli bacteria.
Course and Zab Unit structure. Explain the steps involved in the
transformation of E. coli.
Unit resources. Micro-pipet usage (please don’t wreck them!!!)
Safety: fire extinguisher, shower, gas shut-
Set up Lab book according to guidelines in the off, eye wash station, EMERGENCY.
Resource Manual (pg 90 - 91) and Unit style.
Transform E. coli cells with plasmid DNA. Basic Transformation Flow Chart:
E.coli JM101 cells on pick plate
add to cold calcium chloride
divide into 2 tubes
add TE add plasmid
45 C heat pulse
add LB broth
37 C incubation
When recording a protocol in your lab book—can someone pick up your lab book and do the experiment
exactly the way you did it??? Micropipette Usage: The Gilson (navy blue) pipettors are the main ones we use; all of them have 3 digits
in their display but each means a different volume. When selecting a pipettor, make sure its pipetting range
includes the volume you want to pipet; i.e. if you want to pipet 500 µL, do not use the p200 pipettor since
its range is 50 µL to 200 µL.
Name of pipettor: Range: Colour of tip: Volume example:
P1000 - blue cap 200 µL to 1000 µL blue 050 = 500 µL
P200 - yellow cap 50 µL to 200 µL yellow 050 = 50 µL
P20 - yellow cap 1.0 µL to 20.0 µL yellow 050 = 5.0 µL
We also have Eppendorf pipettors (cream coloured) which have a range of 10.0 µL to 100.0 µL;
e.g. 0500 = 50.0 µL and they take a yellow tip (don’t forget the decimal).
A number of these pipettes have already been destroyed last term—please,
be very careful with these; ask if you are in the least bit unsure about the
number or how to change them. Do not try to dial below or above the range
of the pipettor you are using—you are destroying the pipettor if you do.
None of the pipettors should make any noise when changing the volume on
the dial. Learn what the numbers mean on the different pipettors. Quiz #1
will include a question about the pipettors and pipetting.
Safety: Lab coats, long pants, and glasses required for bench work. NO open-toed shoes. Long-
hair tied back. NO food or drink in the lab.
Beware of hazards associated with Bunsen burners, handling of bacteria etc..
BE AWARE OF WHAT YOU ARE DOING!!!
Background Prep: Be aware of what the numbers mean and the ranges of the micropipettors mentioned
Read the “Practical” file on OWL so you are aware of the skills we will be testing
you in Session 6 and you will be learning in this lab period.
Read the following: Antibiotics (ampicillin and kanamycin), Aseptic technique
(Pouring plates, Transfer techniques, Pipetting tips), Micropipettes, Plasmids, Safety
in the Laboratory, Scientific Nomenclature, and Transformation background material
in Resource Manual.
Assignment(s): “Exit Stamp” for Transformation Protocol in Lab Book. Session 2: Transformation Background
The first experiment will provide context for a discussion of basic principles underlying transformation and
antibiotic resistance in bacteria. The relevance and importance of controls will also be discussed along with
proper data recording procedures.
Most of session 3 is devoted to designing experiments. We will begin that process in this class by
introducing the “Toy Box”.
Explain the relevance and importance of controls in Describe the mechanism of transformation and
experimental designs. Discuss antibiotics and the structure of an R-plasmid
resistance. What is a transformant? What is a
Initiate your Independent Experiment by
Develop a raw data table. discussing the “Toy Box”
Background Prep: Review Antibiotics and Plasmids.
Look at Independent Experimental Design (Antibiotic Physiology, Curing and
Mutagenesis) in the resource manual and the Requisition Form on OWL. Session 3: Experimental Design
We will begin with a short Quiz (2%) which may include questions of micropipette usage, the